CN104988167A - Siraitia grosvenorii swingle cucurbitadienol synthetase gene SgCbQ and applications thereof - Google Patents

Abstract

The present invention discloses a Siraitia grosvenorii swingle cucurbitadienol synthetase gene SgCbQ and applications of a SgCbQ-containing Saccharomyces cerevisiae recombinant expression vector in production of cucurbitadienol through construction of Saccharomyces cerevisiae engineering bacteria. According to the present invention, SgCbQ is excavated from the Siraitia grosvenorii swingle transcriptome database and has a nucleotide coding sequence represented by SEQ ID NO.1 and a polypeptide sequence represented by SEQ ID NO.2, and after the gene is subjected to codon optimization and chemical synthesis, the gene has a nucleotide coding sequence represented by SEQ ID NO.3; with the application of the Siraitia grosvenorii swingle cucurbitadienol synthetase gene to express in Saccharomyces cerevisiae, the cucurbitadienol having the extremely rare contentin nature can be efficiently synthesized; and the gene has important application values in the fields of related plant reforming through the transgene technology to improve the contents of Cucurbitane type triterpenoids including Mogroside.

Description

Grosvenor Momordica cucurbit dienol synthase gene SgCbQ and application thereof

Technical field

The present invention relates to biological technical field, more specifically relate to one from the cucurbit dienol synthase gene in Grosvenor Momordica sgCbQand the application in synthesis cucurbit dienol.

Background technology

Curcurbitaceae liana deciduous plant Grosvenor Momordica ( siraitia grosvenoriiswingle) be the specific precious medicinal and sweetener plant of China, be mainly distributed in the Guangxi province of China, included in Pharmacopoeia of People's Republic of China since 1977 always, use as conventional Chinese medicine.Grosvenor Momordica is listed in first and " is medicine and the kind list of food " by the Ministry of Health and the State Administration of Traditional Chinese Medicines.Arhat fruity is extremely sweet, cool in nature, nontoxic, there is relieving cough and moistening lung, reduce phlegm enrich blood, relax bowel, dispel functional nucleotide sequences such as the stasis of blood (Prakash, Indra, and Venkata Sai Prakash Chaturvedula. Additional New Minor Cucurbitane Glycosides from siraitia grosvenorii. molecules19.3 (2014): 3669-3680.).The sweet glucoside of cucurbitane type tetracyclic triterpenes is the main active substances in Grosvenor Momordica, it is the desirable natural sweeteners of a kind of high sugariness, low calory, there is the effect (Chiu such as hypoglycemic, anti-oxidant, anticancer, antiviral in addition, Chun-Hui, et al. Biotransformation of Mogrosides from siraitia grosvenoriiswingle by Saccharomyces cerevisiae. Journal of agricultural and food chemistry 61.29 (2013): 7127-7134.).

Cucurbit dienol is the common precursor material of all cucurbitane type triterpene compounds comprising Momordica-Glycosides, it is one of modal triterpene compound in plant, it is generated through the reaction of a sequence under the catalysis of cucurbit dienol synthetic enzyme by 2,3-oxidosqualene.Cucurbit dienol synthetic enzyme is rate-limiting enzyme crucial in cucurbitane type triterpene compound route of synthesis, and the clone of this gene and the research of functional verification to cucurbitane type triterpene compound have great importance.

So far, only from Curcurbitaceae cucurbita plants pumpkin, clone obtains a cucurbit dienol synthase gene (Shibuya, Masaaki, Shinya Adachi, and Yutaka Ebizuka. Cucurbitadienol synthase, the first committed enzyme for cucurbitacin biosynthesis, is a distinct enzyme from cycloartenol synthase for phytosterol biosynthesis. Tetrahedron 60.33 (2004): 6995-7003.), this gene is expressed in yeast saccharomyces cerevisiae GIL77 can generate the single product of cucurbit dienol.But for the plant Grosvenor Momordica of a large amount of accumulation cucurbitane type triterpene compound, the cucurbit dienol synthase gene that can synthesize cucurbit dienol have not been reported.

Summary of the invention

An object of the present invention is to provide the cucurbit dienol synthase gene excavated from Grosvenor Momordica transcript profile database sgCbQ, its nucleotide sequence is as SEQ ID NO:1, and coded peptide sequence is as SEQ ID NO:2.Particularly by retrieval Grosvenor Momordica transcript profile database, excavate the gene that an annotation is cucurbit dienol synthetic enzyme sgCbQ, in order to contribute to the expression of this gene in yeast saccharomyces cerevisiae, will sgCbQthe nucleotide sequence after chemosynthesis is carried out as SEQ ID NO:3 after codon optimized.

Two of object of the present invention is to provide the construction process of an Accharomyces cerevisiae GIL77 recombinant bacterial strain.Particularly by the cucurbit dienol synthase gene of chemosynthesis acquisition sgCbQwarp kpni and xhoi double digestion, with same warp kpni and xhothe expression plasmid of yeast pYES2 of I double digestion process connects, and builds recombinant expression plasmid pYES2/SgCbQ.Then by lithium acetate transformation method, recombinant expression plasmid pYES2/SgCbQ is imported in yeast saccharomyces cerevisiae GIL77, build saccharomyces cerevisiae engineered yeast strain GIL77-SgCbQ.

Three of object of the present invention is to provide the synthetic method of cucurbit dienol.Particularly the yeast saccharomyces cerevisiae GIL77-SgCbQ of restructuring is inoculated in 20 mL lack uridylic yeast synthetic medium (supplement with the addition of 20 g/L glucose, 20 mg/L ergosterols, the tween 80 of 13 mg/L protohemines and 5 g/L) in, in 30 DEG C, cultivate 48 h under 200rpm condition, yeast cell is bred in a large number.Then collect yeast cell and transfer in the yeast synthetic medium lacking uridylic and (supplement and with the addition of 20 g/L semi-lactosis, 20 mg/L ergosterols, the tween 80 of 13 mg/L protohemines and 5 g/L) in 30 DEG C, 24 h are cultivated, the expression of induction cucurbit dienol synthetic enzyme under 200rpm condition; Finally collect yeast thalline to lay equal stress on and outstandingly to contain with 20 mL in the substratum of 3% glucose and 13 mg/L protohemines in 30 DEG C, continue cultivation 24 h under 200rpm condition, utilize the cucurbit dienol synthetic enzyme synthesis cucurbit dienol of yeast cylinder accumulation.After collected by centrifugation yeast cell, by saponification reaction Breaking Yeast thalline, the cucurbit dienol product in the broken liquid of recycling n-hexane extraction yeast.

Adopt above technical scheme, the invention provides a kind of cucurbit dienol synthase gene sgCbQand the application in synthesis cucurbit dienol.Experiment proves, cucurbit dienol synthase gene sgCbQcan express in yeast saccharomyces cerevisiae GIL77, product is accredited as cucurbit dienol through GC-MS.Utilize cucurbit dienol synthase gene sgCbQefficiently can synthesize the cucurbit dienol that occurring in nature content is extremely rare, there is important practice significance.Meanwhile, the discovery of this gene also provides important theoretical basis for utilizing genetic engineering bacterium transformation corresponding plants to improve the content comprising the cucurbitane type triterpene compound of Momordica-Glycosides.

Accompanying drawing explanation

Fig. 1 shows Grosvenor Momordica cucurbit dienol synthetic enzyme S gCbQwith pumpkin cpCbQthe comparison result of peptide sequence.

Fig. 2 shows the physical map of constructed recombinant yeast expression vector pYES2-SgCbQ

Fig. 3 shows the gas chromatogram of cucurbit dienol, and (A is the gas chromatogram of the saccharomyces cerevisiae engineered yeast extraction product that have expressed empty carrier pYES2; B is the gas chromatogram of cucurbit dienol standard substance; C is the gas chromatogram of the saccharomyces cerevisiae engineered yeast extraction product that have expressed recombinant plasmid pYES2-SgCbQ)

Fig. 4 shows the mass spectrum of cucurbit dienol, and (A is the mass spectrum of cucurbit dienol standard substance; B is that Yeast engineering bacteria extraction product 16.4 min in gas phase collection of illustrative plates having reached recombinant plasmid pYES2-SgCbQ go out the mass spectrum at peak place).

Embodiment

Below in conjunction with accompanying drawing and concrete case study on implementation, the specific embodiment of the present invention is described in detail.Following embodiment only for illustration of the present invention, but is not used in restriction use range of the present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, several variation and improvement can also be made.These all belong to protection scope of the present invention.Unreceipted specific experiment method in following case study on implementation, usual conveniently condition, the molecular cloning of such as Sambrook etc.: laboratory manual (New York:Cold Spring Harbor Laboratory Pross, 1989) condition described in, or according to the suggestion that related reagent manufacturer provides.

The structure of embodiment 1, yeast saccharomyces cerevisiae recombinant plasmid

The gene that an annotation is cucurbit dienol synthetic enzyme is excavated from Grosvenor Momordica transcript profile database sgCbQ(GeneBank ID NO:HQ128567), this gene and the unique gene with cucurbit dienol synthetic enzyme function reported at present cpCbQthe amino acid identity of (GeneBank ID NO:AB116238) is 88%(accompanying drawing 1), the results presumption of transcript profile and express spectra sgCbQit may be potential cucurbit dienol synthetic enzyme.By analyzing yeast saccharomyces cerevisiae to the use Preference of codon, we are right sgCbQgene carry out codon optimized after will sgCbQgene transfers to Qing Lan bio tech ltd, Wuxi to synthesize, synthesis sgCbQgene fragment order is as shown in SEQ ID NO.3.Then by synthesis sgCbQgene is inserted into the multiple clone site of Yeast expression carrier pYES2 kpni and xhoconstruction recombination plasmid pYES2-SgCbQ between I, sequence such as the SEQ ID NO.4(recombinant plasmid physical map of recombinant plasmid is shown in accompanying drawing 2).

The structure of embodiment 2, yeast saccharomyces cerevisiae recombinant bacterial strain

To verify in embodiment 1 that correct recombinant plasmid pYES2-SgCbQ imports in yeast saccharomyces cerevisiae GIL77 by lithium acetate transformation method, then the yeast saccharomyces cerevisiae GIL77 after conversion is coated the auxotroph lacking uridylic and screen on flat board.Due on recombinant plasmid pYES2-SgCbQ with uridylic encoding gene, the recombinant Saccharomyces cerevisiae GIL77 of successful conversion pYES-SgCbQ plasmid can generate on the flat board lacking uridylic.The restructuring bacterium colony grown is again through again extracting yeast plasmid and verifying through PCR sgCbQafter gene is present in recombinant Saccharomyces cerevisiae GIL77, can be used as follow-up fermentation confirmatory experiment.

The synthesis of embodiment 3, cucurbit dienol

Select case to implement in 2, to verify that the yeast synthetic medium that correct Wine brewing yeast strain list colony inoculation lacks uridylic in 20 mL (supplements and with the addition of 20 g/L glucose, 20 mg/L ergosterols, the tween 80 of 13 mg/L protohemines and 5 g/L) in, in 30 DEG C, under 200rpm condition, cultivate 48 h.Then collecting cell with the deionized water wash three times of sterilizing, (supplement at the yeast synthetic medium lacking uridylic and with the addition of 20 g/L semi-lactosis, 20 mg/L ergosterols, the tween 80 of 13 mg/L protohemines and 5 g/L) in 30 DEG C, induce 24 h under 200rpm condition, impel cucurbit dienol synthetic enzyme great expression to accumulate; Collecting cell again, with the deionized water wash cell three times of sterilizing, and the yeast body weight of collection is hanged in the substratum containing 3% glucose and 13 mg/L protohemines with 20 mL in 30 DEG C, cultivation 24 h is continued under 200rpm condition, yeast can include from health 2,3-oxidosqualene is substrate, utilizes the cucurbit dienol synthetic enzyme synthesis cucurbit dienol of induction period cylinder accumulation.

The extraction of embodiment 4, cucurbit dienol

Collect above-mentioned yeast thalline, with 5 mL 40% KOH/ 50% ethanol (V/V:1:1) saponification 10 min under 85 DEG C of water bath condition.Then with the normal hexane of 5 mL, 3 times are extracted to saponification resultant, merge n-hexane extraction product, and by vacuum rotary steam, concentrate drying is carried out to product.Finally the product of drying is dissolved in 500 μ L normal hexanes, after 0.22 μm of membrane filtration process, utilizes GC-MS to analyze.

Embodiment 5, GC-MS detect cucurbit dienol product

Gas chromatograph is Agilent 7890, and mass spectrograph is Agilent Q-TOF-MS. analytical column is mm × 0.5 μm, HP-5ms(30 m × 0.25).Moving phase is He, and splitting ratio is 100:1, and sample size is 1 μ L, and column oven heating schedule is: 80 DEG C, 1min; 20 DEG C/min rises to 300 DEG C; 300 DEG C maintain 15 min.Mass spectrum is ESI source, positive ion mode.

Gas chromatograph results (accompanying drawing 3) display with sgCbQyeast recombinant strain strain extract in generate a unique new product peak at 16.40 min places, this product peak is consistent with the appearance time of cucurbit dienol standard substance, and the negative control bacterial strain having transformed empty carrier does not have product peak at 16.4 min places.Further mass spectral results display (accompanying drawing 4), with sgCbQyeast recombinant strain strain go out the Information in Mass Spectra of the product at peak place and the Information in Mass Spectra of cucurbit dienol standard substance is on all four (m/z at gas-chromatography 16.4 min, 119 [100], 134 [99], 259 [75], 274 [71], 121 [70], 95 [58], 107 [55], 207 [52], 161 [51], 81 [44], 136 [36], 69 [36], 149 [28], 175 [23], 408 [16], 393 [14], 231 [13], 55 [6], 411 [M-CH ₃] ⁺[4], 426 [M] ⁺[3]).These data show, sgCbQit is the cucurbit dienol synthetic enzyme with cucurbit dienol complex functionality.

Above specific embodiment of the invention case is described.Use the cucurbit dienol synthase gene found in the present invention can synthesize the very rare cucurbit dienol of occurring in nature content, and fermentation condition is simple, the separation and purification of product is easy, can realize a large amount of synthesis of cucurbit dienol in yeast.It is to be appreciated that the present invention is not limited to particular implementation described above, those skilled in the art can make various distortion or amendment in the atmosphere of claim, and this does not affect flesh and blood of the present invention.

SEQUENCE LISTING

 

<110> Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences

 

<120> Grosvenor Momordica cucurbit dienol synthase gene SgCbQ and application thereof

 

<130> 1

 

<160> 4

 

<170> PatentIn version 3.3

 

<210> 1

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<212> DNA

<213> Grosvenor Momordica

 

<400> 1

atgtggaggt taaaggtcgg agcagaaagc gttggggaga atgatgagaa atggttgaag 60

 

agcataagca atcacttggg acgccaggtg tgggagttct gtccggatgc cggcacccaa 120

 

caacagctct tgcaagtcca caaagctcgt aaagctttcc acgatgaccg tttccaccga 180

 

aagcaatctt ccgatctctt tatcactatt cagtatggaa aggaagtaga aaatggtgga 240

 

aagacagcgg gagtgaaatt gaaagaaggg gaagaggtga ggaaagaggc agtagagagt 300

 

agcttagaga gggcattaag tttctactca agcatccaga caagcgatgg gaactgggct 360

 

tcggatcttg gggggcccat gtttttactt ccgggtctgg tgattgccct ctacgttaca 420

 

ggcgtcttga attctgtttt atccaagcac caccggcaag agatgtgcag atatgtttac 480

 

aatcaccaga atgaagatgg ggggtggggt ctccacatcg agggcccaag caccatgttt 540

 

ggttccgcac tgaattatgt tgcactcagg ctgcttggag aagacgccaa cgccggggca 600

 

atgccaaaag cacgtgcttg gatcttggac cacggtggcg ccaccggaat cacttcctgg 660

 

ggcaaattgt ggctttctgt acttggagtc tacgaatgga gtggcaataa tcctcttcca 720

 

cccgaatttt ggttatttcc ttacttccta ccatttcatc caggaagaat gtggtgccat 780

 

tgtcgaatgg tttatctacc aatgtcatac ttatatggaa agagatttgt tgggccaatc 840

 

acacccatag ttctgtctct cagaaaagaa ctctacgcag ttccatatca tgaaatagac 900

 

tggaataaat ctcgcaatac atgtgcaaag gaggatctgt actatccaca tcccaagatg 960

 

caagatattc tgtggggatc tctccaccac gtgtatgagc ccttgtttac tcgttggcct 1020

 

gccaaacgcc tgagagaaaa ggctttgcag actgcaatgc aacatattca ctatgaagat 1080

 

gagaataccc gatatatatg ccttggccct gtcaacaagg tactcaatct gctttgttgt 1140

 

tgggttgaag atccctactc cgacgccttc aaacttcatc ttcaacgagt ccatgactat 1200

 

ctctgggttg ctgaagatgg catgaaaatg cagggttata atgggagcca gttgtgggac 1260

 

actgctttct ccatccaagc aatcgtatcc accaaacttg tagacaacta tggcccaacc 1320

 

ttaagaaagg cacacgactt cgttaaaagt tctcagattc agcaggactg tcctggggat 1380

 

cctaatgttt ggtaccgtca cattcataaa ggtgcatggc cattttcaac tcgagatcat 1440

 

ggatggctca tctctgactg tacagcagag ggattaaagg ctgctttgat gttatccaaa 1500

 

cttccatccg aaacagttgg ggaatcatta gaacggaatc gcctttgcga tgctgtaaac 1560

 

gttctccttt ctttgcaaaa cgataatggt ggctttgcat catatgagtt gacaagatca 1620

 

tacccttggt tggagttgat caaccccgca gaaacgtttg gagatattgt cattgattat 1680

 

ccgtatgtgg agtgcacctc agccacaatg gaagcactga cgttgtttaa gaaattacat 1740

 

cccggccata ggaccaaaga aattgatact gctattgtca gggcggccaa cttccttgaa 1800

 

aatatgcaaa ggacggatgg ctcttggtat ggatgttggg gggtttgctt cacgtatgcg 1860

 

gggtggtttg gcataaaggg attggtggct gcaggaagga catataataa ttgccttgcc 1920

 

attcgcaagg cttgcgattt tttactatct aaagagctgc ccggcggtgg atggggagag 1980

 

agttaccttt catgtcagaa taaggtatac acaaatcttg aaggaaacag accgcacctg 2040

 

gttaacacgg cctgggtttt aatggccctc atagaagctg gccaggctga gagagaccca 2100

 

acaccattgc atcgtgcagc aaggttgtta atcaattccc agttggagaa tggtgatttc 2160

 

ccccaacagg agatcatggg agtctttaat aaaaattgca tgatcacata tgctgcatac 2220

 

cgaaacattt ttcccatttg ggctcttgga gagtattgcc atcgggtttt gactgaataa 2280

 

 

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Met Trp Arg Leu Lys Val Gly Ala Glu Ser Val Gly Glu Asn Asp Glu

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Lys Trp Leu Lys Ser Ile Ser Asn His Leu Gly Arg Gln Val Trp Glu

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Phe Cys Pro Asp Ala Gly Thr Gln Gln Gln Leu Leu Gln Val His Lys

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Ala Arg Lys Ala Phe His Asp Asp Arg Phe His Arg Lys Gln Ser Ser

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Asp Leu Phe Ile Thr Ile Gln Tyr Gly Lys Glu Val Glu Asn Gly Gly

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Lys Thr Ala Gly Val Lys Leu Lys Glu Gly Glu Glu Val Arg Lys Glu

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Ala Val Glu Ser Ser Leu Glu Arg Ala Leu Ser Phe Tyr Ser Ser Ile

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Gln Thr Ser Asp Gly Asn Trp Ala Ser Asp Leu Gly Gly Pro Met Phe

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Leu Leu Pro Gly Leu Val Ile Ala Leu Tyr Val Thr Gly Val Leu Asn

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Ser Val Leu Ser Lys His His Arg Gln Glu Met Cys Arg Tyr Val Tyr

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Asn His Gln Asn Glu Asp Gly Gly Trp Gly Leu His Ile Glu Gly Pro

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Ser Thr Met Phe Gly Ser Ala Leu Asn Tyr Val Ala Leu Arg Leu Leu

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Gly Glu Asp Ala Asn Ala Gly Ala Met Pro Lys Ala Arg Ala Trp Ile

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Leu Asp His Gly Gly Ala Thr Gly Ile Thr Ser Trp Gly Lys Leu Trp

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Leu Ser Val Leu Gly Val Tyr Glu Trp Ser Gly Asn Asn Pro Leu Pro

225 230 235 240

 

 

Pro Glu Phe Trp Leu Phe Pro Tyr Phe Leu Pro Phe His Pro Gly Arg

245 250 255

 

 

Met Trp Cys His Cys Arg Met Val Tyr Leu Pro Met Ser Tyr Leu Tyr

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Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Ile Val Leu Ser Leu Arg

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Lys Glu Leu Tyr Ala Val Pro Tyr His Glu Ile Asp Trp Asn Lys Ser

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Arg Asn Thr Cys Ala Lys Glu Asp Leu Tyr Tyr Pro His Pro Lys Met

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Gln Asp Ile Leu Trp Gly Ser Leu His His Val Tyr Glu Pro Leu Phe

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Thr Arg Trp Pro Ala Lys Arg Leu Arg Glu Lys Ala Leu Gln Thr Ala

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Met Gln His Ile His Tyr Glu Asp Glu Asn Thr Arg Tyr Ile Cys Leu

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Gly Pro Val Asn Lys Val Leu Asn Leu Leu Cys Cys Trp Val Glu Asp

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Pro Tyr Ser Asp Ala Phe Lys Leu His Leu Gln Arg Val His Asp Tyr

385 390 395 400

 

 

Leu Trp Val Ala Glu Asp Gly Met Lys Met Gln Gly Tyr Asn Gly Ser

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Gln Leu Trp Asp Thr Ala Phe Ser Ile Gln Ala Ile Val Ser Thr Lys

420 425 430

 

 

Leu Val Asp Asn Tyr Gly Pro Thr Leu Arg Lys Ala His Asp Phe Val

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Lys Ser Ser Gln Ile Gln Gln Asp Cys Pro Gly Asp Pro Asn Val Trp

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Tyr Arg His Ile His Lys Gly Ala Trp Pro Phe Ser Thr Arg Asp His

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Gly Trp Leu Ile Ser Asp Cys Thr Ala Glu Gly Leu Lys Ala Ala Leu

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Met Leu Ser Lys Leu Pro Ser Glu Thr Val Gly Glu Ser Leu Glu Arg

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Asn Arg Leu Cys Asp Ala Val Asn Val Leu Leu Ser Leu Gln Asn Asp

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Asn Gly Gly Phe Ala Ser Tyr Glu Leu Thr Arg Ser Tyr Pro Trp Leu

530 535 540

 

 

Glu Leu Ile Asn Pro Ala Glu Thr Phe Gly Asp Ile Val Ile Asp Tyr

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Pro Tyr Val Glu Cys Thr Ser Ala Thr Met Glu Ala Leu Thr Leu Phe

565 570 575

 

 

Lys Lys Leu His Pro Gly His Arg Thr Lys Glu Ile Asp Thr Ala Ile

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Val Arg Ala Ala Asn Phe Leu Glu Asn Met Gln Arg Thr Asp Gly Ser

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Trp Tyr Gly Cys Trp Gly Val Cys Phe Thr Tyr Ala Gly Trp Phe Gly

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Ile Lys Gly Leu Val Ala Ala Gly Arg Thr Tyr Asn Asn Cys Leu Ala

625 630 635 640

 

 

Ile Arg Lys Ala Cys Asp Phe Leu Leu Ser Lys Glu Leu Pro Gly Gly

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Gly Trp Gly Glu Ser Tyr Leu Ser Cys Gln Asn Lys Val Tyr Thr Asn

660 665 670

 

 

Leu Glu Gly Asn Arg Pro His Leu Val Asn Thr Ala Trp Val Leu Met

675 680 685

 

 

Ala Leu Ile Glu Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Leu His

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Arg Ala Ala Arg Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe

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Pro Gln Gln Glu Ile Met Gly Val Phe Asn Lys Asn Cys Met Ile Thr

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Tyr Ala Ala Tyr Arg Asn Ile Phe Pro Ile Trp Ala Leu Gly Glu Tyr

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Cys His Arg Val Leu Thr Glu

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caacaattgt tgcaagttca caaggctaga aaggctttcc acgacgacag attccacaga 180

 

aagcaatctt ctgacttgtt catcactatc caatacggta aggaagttga aaacggtggt 240

 

aagactgctg gtgttaagtt gaaggaaggt gaagaagtta gaaaggaagc tgttgaatct 300

 

tctttggaaa gagctttgtc tttctactct tctatccaaa cttctgacgg taactgggct 360

 

tctgacttgg gtggtccaat gttcttgttg ccaggtttgg ttatcgcttt gtacgttact 420

 

ggtgttttga actctgtttt gtctaagcac cacagacaag aaatgtgtag atacgtttac 480

 

aaccaccaaa acgaagacgg tggttggggt ttgcacatcg aaggtccatc tactatgttc 540

 

ggttctgctt tgaactacgt tgctttgaga ttgttgggtg aagacgctaa cgctggtgct 600

 

atgccaaagg ctagagcttg gatcttggac cacggtggtg ctactggtat cacttcttgg 660

 

ggtaagttgt ggttgtctgt tttgggtgtt tacgaatggt ctggtaacaa cccattgcca 720

 

ccagaattct ggttgttccc atacttcttg ccattccacc caggtagaat gtggtgtcac 780

 

tgtagaatgg tttacttgcc aatgtcttac ttgtacggta agagattcgt tggtccaatc 840

 

actccaatcg ttttgtcttt gagaaaggaa ttgtacgctg ttccatacca cgaaatcgac 900

 

tggaacaagt ctagaaacac ttgtgctaag gaagacttgt actacccaca cccaaagatg 960

 

caagacatct tgtggggttc tttgcaccac gtttacgaac cattgttcac tagatggcca 1020

 

gctaagagat tgagagaaaa ggctttgcaa actgctatgc aacacatcca ctacgaagac 1080

 

gaaaacacta gatacatctg tttgggtcca gttaacaagg ttttgaactt gttgtgttgt 1140

 

tgggttgaag acccatactc tgacgctttc aagttgcact tgcaaagagt tcacgactac 1200

 

ttgtgggttg ctgaagacgg tatgaagatg caaggttaca acggttctca attgtgggac 1260

 

actgctttct ctatccaagc tatcgtttct actaagttgg ttgacaacta cggtccaact 1320

 

ttgagaaagg ctcacgactt cgttaagtct tctcaaatcc aacaagactg tccaggtgac 1380

 

ccaaacgttt ggtacagaca catccacaag ggtgcttggc cattctctac tagagaccac 1440

 

ggttggttga tctctgactg tactgctgaa ggtttgaagg ctgctttgat gttgtctaag 1500

 

ttgccatctg aaactgttgg tgaatctttg gaaagaaaca gattgtgtga cgctgttaac 1560

 

gttttgttgt ctttgcaaaa cgacaacggt ggtttcgctt cttacgaatt gactagatct 1620

 

tacccatggt tggaattgat caacccagct gaaactttcg gtgacatcgt tatcgactac 1680

 

ccatacgttg aatgtacttc tgctactatg gaagctttga ctttgttcaa gaagttgcac 1740

 

ccaggtcaca gaactaagga aatcgacact gctatcgtta gagctgctaa cttcttggaa 1800

 

aacatgcaaa gaactgacgg ttcttggtac ggttgttggg gtgtttgttt cacttacgct 1860

 

ggttggttcg gtatcaaggg tttggttgct gctggtagaa cttacaacaa ctgtttggct 1920

 

atcagaaagg cttgtgactt cttgttgtct aaggaattgc caggtggtgg ttggggtgaa 1980

 

tcttacttgt cttgtcaaaa caaggtttac actaacttgg aaggtaacag accacacttg 2040

 

gttaacactg cttgggtttt gatggctttg atcgaagctg gtcaagctga aagagaccca 2100

 

actccattgc acagagctgc tagattgttg atcaactctc aattggaaaa cggtgacttc 2160

 

ccacaacaag aaatcatggg tgttttcaac aagaactgta tgatcactta cgctgcttac 2220

 

agaaacatct tcccaatctg ggctttgggt gaatactgtc acagagtttt gactgaataa 2280

 

 

<210> 4

<211> 8067

<212> DNA

<213> artificial sequence

 

<400> 4

ggccgcaaat taaagccttc gagcgtccca aaaccttctc aagcaaggtt ttcagtataa 60

 

tgttacatgc gtacacgcgt ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg 120

 

ttcttaatac taacataact ataaaaaaat aaatagggac ctagacttca ggttgtctaa 180

 

ctccttcctt ttcggttaga gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa 240

 

ctaattacat gatgcggccc tctagatgca tgctcgagtt attcagtcaa aactctgtga 300

 

cagtattcac ccaaagccca gattgggaag atgtttctgt aagcagcgta agtgatcata 360

 

cagttcttgt tgaaaacacc catgatttct tgttgtggga agtcaccgtt ttccaattga 420

 

gagttgatca acaatctagc agctctgtgc aatggagttg ggtctctttc agcttgacca 480

 

gcttcgatca aagccatcaa aacccaagca gtgttaacca agtgtggtct gttaccttcc 540

 

aagttagtgt aaaccttgtt ttgacaagac aagtaagatt caccccaacc accacctggc 600

 

aattccttag acaacaagaa gtcacaagcc tttctgatag ccaaacagtt gttgtaagtt 660

 

ctaccagcag caaccaaacc cttgataccg aaccaaccag cgtaagtgaa acaaacaccc 720

 

caacaaccgt accaagaacc gtcagttctt tgcatgtttt ccaagaagtt agcagctcta 780

 

acgatagcag tgtcgatttc cttagttctg tgacctgggt gcaacttctt gaacaaagtc 840

 

aaagcttcca tagtagcaga agtacattca acgtatgggt agtcgataac gatgtcaccg 900

 

aaagtttcag ctgggttgat caattccaac catgggtaag atctagtcaa ttcgtaagaa 960

 

gcgaaaccac cgttgtcgtt ttgcaaagac aacaaaacgt taacagcgtc acacaatctg 1020

 

tttctttcca aagattcacc aacagtttca gatggcaact tagacaacat caaagcagcc 1080

 

ttcaaacctt cagcagtaca gtcagagatc aaccaaccgt ggtctctagt agagaatggc 1140

 

caagcaccct tgtggatgtg tctgtaccaa acgtttgggt cacctggaca gtcttgttgg 1200

 

atttgagaag acttaacgaa gtcgtgagcc tttctcaaag ttggaccgta gttgtcaacc 1260

 

aacttagtag aaacgatagc ttggatagag aaagcagtgt cccacaattg agaaccgttg 1320

 

taaccttgca tcttcatacc gtcttcagca acccacaagt agtcgtgaac tctttgcaag 1380

 

tgcaacttga aagcgtcaga gtatgggtct tcaacccaac aacacaacaa gttcaaaacc 1440

 

ttgttaactg gacccaaaca gatgtatcta gtgttttcgt cttcgtagtg gatgtgttgc 1500

 

atagcagttt gcaaagcctt ttctctcaat ctcttagctg gccatctagt gaacaatggt 1560

 

tcgtaaacgt ggtgcaaaga accccacaag atgtcttgca tctttgggtg tgggtagtac 1620

 

aagtcttcct tagcacaagt gtttctagac ttgttccagt cgatttcgtg gtatggaaca 1680

 

gcgtacaatt cctttctcaa agacaaaacg attggagtga ttggaccaac gaatctctta 1740

 

ccgtacaagt aagacattgg caagtaaacc attctacagt gacaccacat tctacctggg 1800

 

tggaatggca agaagtatgg gaacaaccag aattctggtg gcaatgggtt gttaccagac 1860

 

cattcgtaaa cacccaaaac agacaaccac aacttacccc aagaagtgat accagtagca 1920

 

ccaccgtggt ccaagatcca agctctagcc tttggcatag caccagcgtt agcgtcttca 1980

 

cccaacaatc tcaaagcaac gtagttcaaa gcagaaccga acatagtaga tggaccttcg 2040

 

atgtgcaaac cccaaccacc gtcttcgttt tggtggttgt aaacgtatct acacatttct 2100

 

tgtctgtggt gcttagacaa aacagagttc aaaacaccag taacgtacaa agcgataacc 2160

 

aaacctggca acaagaacat tggaccaccc aagtcagaag cccagttacc gtcagaagtt 2220

 

tggatagaag agtagaaaga caaagctctt tccaaagaag attcaacagc ttcctttcta 2280

 

acttcttcac cttccttcaa cttaacacca gcagtcttac caccgttttc aacttcctta 2340

 

ccgtattgga tagtgatgaa caagtcagaa gattgctttc tgtggaatct gtcgtcgtgg 2400

 

aaagcctttc tagccttgtg aacttgcaac aattgttgtt gagtaccagc gtctggacag 2460

 

aattcccaaa cttgtctacc caagtggtta gagatagact tcaaccactt ttcgtcgttt 2520

 

tcaccaacag attcagcacc aaccttcaat ctccacatgg taccaagctt aatattccct 2580

 

atagtgagtc gtattacagc tgctagtagt ccgatccggg gttttttctc cttgacgtta 2640

 

aagtatagag gtatattaac aattttttgt tgatactttt attacatttg aataagaagt 2700

 

aatacaaacc gaaaatgttg aaagtattag ttaaagtggt taatgcagtt tttgcattta 2760

 

tatatctgtt aatagatcaa aaatcatcgc ttcgctgatt aattacccca gaaataaggc 2820

 

taaaaaacta atcgcattat catcctatgg ttgttaattt gattcgttca tttgaaggtt 2880

 

tgtggggcca ggttactgcc aatttttcct cttcataacc ataaaagcta gtattgtaga 2940

 

atctttattg ttcggagcag tgcggcgcga ggcacatctg cgtttcagga acgcgaccgg 3000

 

tgaggacgag gacgcacgga ggagagtctt ccttcggagg gctgtcaccc gctcggcggc 3060

 

ttctaatccg tactagtgga tcatccccac gcgccctgta gcgccccatt aagcgcggcg 3120

 

ggtgtggtgg ttacgcccag cgtgacccct acacttccca ccgccctagc ccccgctcct 3180

 

ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 3240

 

gggggcatcc gtttaccctt ccgatttact gctttacggc acctcgaccc caaaaaactt 3300

 

gattagggtg atggttcacg tagtgggcca tcgccctgat agaccctttt tcgccctttg 3360

 

acgttggagt ccacgttctt taatagtgga ctcttgttgg aaactggaac aacactcaac 3420

 

cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggg ctattcgtta 3480

 

aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca 3540

 

atttaaatat ttgcttatac aatcttcctg tttttggggc ttttctgatt atcaaccggg 3600

 

gtggagcttc ccattgcgaa taccgcttcc acaaacattg ctcaaaagta tctctttgct 3660

 

atatatctct gtgctatatc cctatataac ctacccatcc acctttcgct ccttgaactt 3720

 

gcatctaaac tcgacctcta cattttttat gtttatctct agtattactc tttagacaaa 3780

 

aaaattgtag taagaactat tcatagagtg aatcgaaaac aatacgaaaa tgtaaacatt 3840

 

tcctatacgt agtatataga gacaaaatag aagaaaccgt tcataatttt ctgaccaatg 3900

 

aagaatcatc aacgctatca ctttctgttc acaaagtatg cgcaatccac atcggtatag 3960

 

aatataatcg gggatgcctt tatcttgaaa aaatgcaccc gcagcttcgc tagtaatcag 4020

 

taaacgcggg aagtggagtc aggctttttt tatggaagag aaaatagaca ccaaagtagc 4080

 

cttcttctaa ccttaacgga cctacagtgc aaaaagttat caagagactg cattatagag 4140

 

cgcacaaagg agaaaaaaag taatctaaga tgctttgtta gaaaaatagc gctctcggga 4200

 

tgcatttttg tagaacaaaa aagaagtata gattctttgt tggtaaaata gcgctctcgc 4260

 

gttgcatttc tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa ttagcgctct 4320

 

cgtcgcgttg catttttgtt ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc 4380

 

gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt 4440

 

agcgctctcg cgttgcattt ttgttctaca aaatgaagca cagatgcttc gttaacaaag 4500

 

atatgctatt gaagtgcaag atggaaacgc agaaaatgaa ccggggatgc gacgtgcaag 4560

 

attacctatg caatagatgc aatagtttct ccaggaaccg aaatacatac attgtcttcc 4620

 

gtaaagcgct agactatata ttattataca ggttcaaata tactatctgt ttcagggaaa 4680

 

actcccaggt tcggatgttc aaaattcaat gatgggtaac aagtacgatc gtaaatctgt 4740

 

aaaacagttt gtcggatatt aggctgtatc tcctcaaagc gtattcgaat atcattgaga 4800

 

agctgcagcg tcacatcgga taataatgat ggcagccatt gtagaagtgc cttttgcatt 4860

 

tctagtctct ttctcggtct agctagtttt actacatcgc gaagatagaa tcttagatca 4920

 

cactgccttt gctgagctgg atcaatagag taacaaaaga gtggtaaggc ctcgttaaag 4980

 

gacaaggacc tgagcggaag tgtatcgtac agtagacgga gtatctagta tagtctatag 5040

 

tccgtggaat taattctcat ctttgacagc ttatcatcga taagctagct tttcaattca 5100

 

attcatcatt ttttttttat tctttttttt gatttcggtt tctttgaaat ttttttgatt 5160

 

cggtaatctc cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata 5220

 

tacgcatatg tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca 5280

 

gaacaaaaac ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg 5340

 

ctgctactca tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa 5400

 

caaacttgtg tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag 5460

 

cattaggtcc caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca 5520

 

tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg 5580

 

aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat 5640

 

acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg 5700

 

ttagcggttt gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt 5760

 

tagcagaatt gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg 5820

 

acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg 5880

 

gaagagatga aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg 5940

 

gagacgcatt gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca 6000

 

ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac 6060

 

gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa 6120

 

ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat 6180

 

atcagttatt acccattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 6240

 

tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 6300

 

agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 6360

 

cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 6420

 

taaagttctg ctatgtgata cactattatc ccgtattgac gccgggcaag agcaactcgg 6480

 

tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 6540

 

tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 6600

 

cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 6660

 

gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 6720

 

cataccaaac gacgagagtg acaccacgat gcctgtagca atgccaacaa cgttgcgcaa 6780

 

actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actgaatgga 6840

 

ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 6900

 

tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 6960

 

tggtaagcgc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 7020

 

acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 7080

 

ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 7140

 

ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 7200

 

ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 7260

 

gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 7320

 

ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 7380

 

aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 7440

 

gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 7500

 

gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 7560

 

aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 7620

 

cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 7680

 

tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 7740

 

ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 7800

 

atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 7860

 

cctgggcttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 7920

 

ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 7980

 

gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 8040

 

cgcgcgttgg ccgattcatt aatgcag 8067

 

 

Claims (8)

1. a nucleotide sequence, is characterized in that described nucleotide sequence is from lower group:

there is the nucleotide sequence as shown in SEQ ID NO.1;

but the nucleotide sequence of encode peptide sequence SEQ ID NO.2 shown in different from the nucleotide sequence shown in SEQ ID NO.1, the sequence SEQ ID NO.3 that the nucleotide sequence as shown in SEQ ID NO.1 obtains after codon optimized;

with the Yuan≤90%(of Xu row Tong shown in sequence SEQ ID NO.1 preferably 95%), and coding has the nucleotide sequence of cucurbit dienol synthase activity;

the nucleotide sequence of the sequence hybridization that can limit with sequence table SEQ ID NO.1 under high high stringency conditions

with the nucleotide sequence of arbitrary described nucleotide sequence complementary.

2. a peptide sequence, is characterized in that described polypeptide is selected from lower group:

there is the peptide sequence as shown in SEQ ID NO.2;

by shown in SEQ ID NO.2 peptide sequence through replacement, lack or add one or several amino acid and have cucurbit dienol synthase activity by derivative peptide sequence;

tong Yuan≤the 90%(of peptide sequence shown in peptide sequence and SEQ ID NO.2 preferably 95%), and have synthesis cucurbit dienol activity by derivative peptide sequence.

3. a recombinant expression vector, is characterized in that this expression vector contains nucleotide sequence according to claim 1.

4., according to the recombinant expression vector described in right 3, it is characterized in that it is pYES2-SgCbQ.

5. a genetically engineered host cell, is characterized in that it is the host cell and the progeny cell thereof that transform, expression vector described in right of having transduceed 3 or genome incorporate nucleotide sequence described in right 1.

6., according to the host cell described in right 5, it is characterized in that described host cell is the offspring of bacterial cell, fungal cell, zooblast or vegetable cell and these host cells.

7., according to the host cell described in right 6, it is characterized in that described host cell is Wine brewing yeast strain GIL77.

8. the nucleotide sequence described in right 1, amino acid residue sequence according to claim 2 and the recombinant expression vector described in right 3 are expressed at yeast saccharomyces cerevisiae GIL77 with the application of synthesizing cucurbit dienol.